EP2718436B1 - Materialien und verfahren zur behandlung von pten-mutiertem oder -defizientem krebs - Google Patents

Materialien und verfahren zur behandlung von pten-mutiertem oder -defizientem krebs Download PDF

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EP2718436B1
EP2718436B1 EP12728113.7A EP12728113A EP2718436B1 EP 2718436 B1 EP2718436 B1 EP 2718436B1 EP 12728113 A EP12728113 A EP 12728113A EP 2718436 B1 EP2718436 B1 EP 2718436B1
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inhibitor
pten
cancer
protein kinase
candidate
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EP2718436A1 (de
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Alan Ashworth
Christopher James LORD
Rachel BROUGH
Jessica FRANKUM
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Institute of Cancer Research
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to materials and methods for treating PTEN mutated or deficient cancer using mitotic kinase inhibitors, and to methods of screening for agents for treating PTEN mutated or deficient cancer.
  • SSL synthetic sickness/lethality
  • WO 2012/000103 relates to targeting cancers with PTEN mutations with PLK4 antagonists.
  • Harrington et al. (Nature Medicine, 10(3): 262-267, 2004 ) describes VX-680, a small molecule inhibitor of Aurora kinases.
  • Song et al. (Cell, 144: 187-199, 2011 ) relates to PLK1 and Aurora kinases as targets for inhibition in the treatment of PTEN mutated cancer.
  • WO2010/007756 describes a series of compounds for inhibiting TTK for the treatment of cancer.
  • US2011/0002923 describes the use of a TTK antagonists for treating HER-2 positive breast cancer specifically.
  • US2003/0045491 describes a method for reducing growth of a cancerous cell by contacting it with an agent to reduce TTK activity. There is no mention of the use of TTK inhibitors in the treatment of PTEN mutated or deficient cancers in these documents.
  • the present invention is based on work which attempts to comprehensively define the genetic dependencies for a set of potentially “druggable” genes in a wide range of tumor cell line models. In doing so, we not only reaffirm the impact of PI3-kinase and ERBB2 signalling in the breast cancer but importantly identify novel genetic dependencies for genetically defined subsets of cancer.
  • the present invention is based on the experiments described herein in which inhibition of a mitotic kinase, such as TTK protein kinase, is effective for the treatment of PTEN mutated or deficient cancer.
  • Phosphatase and tensin homolog is a gene that has been identified as a tumor suppressor through the action of its phosphatase product and is mutated in a large number of cancers at high frequency.
  • the protein encoded this gene is a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase. It contains a tensin like domain as well as a catalytic domain similar to that of the dual specificity protein tyrosine phosphatases. Unlike most of the protein tyrosine phosphatases, this protein preferentially dephosphorylates phosphoinositide substrates.
  • the mitotic kinase that may be targeted in all aspects of the present is TTK. set out in the following table: Query Gene ID Symbol Name UniProt ID 1 AURKA ENSG00000087586 AURKA Serine/threonine-protein kinase 6 (EC 2.7.11.1)(Aurora kinase A) (Aurora-A) (Serine/threonine kinase 15)(Aurora/IPL1-related kinase 1)(Aurora-related kinase 1) (hARK1) (Breast tumor-amplified kinase) A3KFJ2 HUMAN 2 TTK ENSG00000112742 TTK Dual specificity protein kinase TTK (EC 2.7.12.1) (Phosphotyrosine picked threonine-protein kinase) (PYT) A8K8U5_HUMAN 3 CDK4 ENSG00000135446 CDK4 Cell division protein kina
  • TTK protein kinase A preferred example of a mitotic kinase, inhibitors of which can be used in accordance with the present invention for the treatment of PTEN mutated or deficient cancer, is TTK protein kinase.
  • This kinase is a dual specificity protein kinase with the ability to phosphorylate tyrosine, serine and threonine.
  • TTK is associated with cell proliferation and is essential for chromosome alignment at the centromere during mitosis and is required for centrosome duplication. It has been found to be a critical mitotic checkpoint protein for accurate segregation of chromosomes during mitosis.
  • the present invention provides an inhibitor of TTK protein kinase for use in a method of treating an individual having cancer by inhibiting said TTK protein kinase, wherein the cancer is a Phosphatase and Tensin Homolog (PTEN) mutated or deficient cancer.
  • the cancer is a Phosphatase and Tensin Homolog (PTEN) mutated or deficient cancer.
  • the present invention provides a method of screening for agents useful in the treatment of a PTEN mutated or deficient cancer, the method employing first and second cell lines, wherein the first cell line is PTEN and the second cell line is PTEN proficient, the method comprising:
  • the present invention provides a method of screening for agents useful in the treatment of PTEN mutated or deficient cancer, the method comprising:
  • the present invention provides a method which comprises having identified a candidate agent useful for the treatment of a PTEN mutated or deficient cancer according to a method as described herein, the further step of manufacturing the compound in bulk and/or formulating the agent in a pharmaceutical composition.
  • TTK inhibitors include relatively potent and orally bioavailable small molecules (PMID: 20383151) (PMID: 21159646).
  • CIN chromosome instability
  • PMID: 21159646 cancer types
  • SAC spindle assembly check point
  • TTK inhibitors are disclosed in WO2010/007756 , US2011/0002923 and US2003/0045491 . These may be suitable for use in accordance with the present invention even though the respective applications do not disclose the use of these TTK inhibitors in the treatment of PTEN mutated or deficient cancers.
  • the inhibitors include compounds AZ3146 or CCT132774 used in the examples below.
  • the present invention also extends to the use of small molecule inhibitors found in the screening disclosed herein and to Derivatives which are compounds of similar structure and functionality to the compounds found in the high throughput screen, but with one or more modifications, are expected to have similar physiological effects to these compounds and could therefore also be of use in the treatment of PTEN mutated or deficient cancer.
  • the screening methods of the invention may be used to screen libraries of such derivatives to optimise their activity, if necessary.
  • Derivatives may be designed, based on a lead compound, by modifying one or more substituents or functional groups compared to the lead compound, for example by replacing these with alternative substituents or groups which are expected to have the same or improved physiological effect.
  • the use of derivatives having such modifications is well known to those in the art.
  • Antibodies may be employed in the present invention as an example of a class of inhibitor useful for treating PTEN mutated or deficient cancer, and more particularly as mitotic kinase inhibitors such as inhibitors of TTK protein kinase. They may also be used in the methods disclosed herein for assessing an individual having cancer or predicting the response of an individual having cancer, in particular for determining whether the individual has PTEN mutated or deficient cancer that might be treatable according to the present invention.
  • the term "antibody” includes an immunoglobulin whether natural or partly or wholly synthetically produced.
  • the term also covers any polypeptide or protein comprising an antibody binding domain.
  • Antibody fragments which comprise an antigen binding domain are such as Fab, scFv, Fv, dAb, Fd; and diabodies. It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP 0 184 187 A , GB 2,188,638 A or EP 0 239 400 A .
  • Antibodies can be modified in a number of ways and the term "antibody molecule" should be construed as covering any specific binding member or substance having an antibody antigen-binding domain with the required specificity. Thus, this term covers antibody fragments and derivatives, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP 0 120 694 A and EP 0 125 023 A .
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment ( Ward, E.S.
  • Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains ( Reiter et al, Nature Biotech, 14: 1239-1245, 1996 ).
  • Minibodies comprising a scFv joined to a CH3 domain may also be made ( Hu et al, Cancer Res., 56: 3055-3061, 1996 ).
  • Preferred antibodies used in accordance with the present invention are isolated, in the sense of being free from contaminants such as antibodies able to bind other polypeptides and/or free of serum components. Monoclonal antibodies are preferred for some purposes, though polyclonal antibodies are within the scope of the present invention.
  • the reactivities of antibodies on a sample may be determined by any appropriate means. Tagging with individual reporter molecules is one possibility.
  • the reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals.
  • the linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non-covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule.
  • One favoured mode is by covalent linkage of each antibody with an individual fluorochrome, phosphor or laser exciting dye with spectrally isolated absorption or emission characteristics.
  • Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red.
  • Suitable chromogenic dyes include diaminobenzidine.
  • Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded.
  • These molecules may be enzymes which catalyse reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed.
  • Antibodies according to the present invention may be used in screening for the presence of a polypeptide, for example in a test sample containing cells or cell lysate as discussed, and may be used in purifying and/or isolating a polypeptide according to the present invention, for instance following production of the polypeptide by expression from encoding nucleic acid. Antibodies may modulate the activity of the polypeptide to which they bind and so, if that polypeptide has a deleterious effect in an individual, may be useful in a therapeutic context (which may include prophylaxis).
  • Peptide fragments that interfere with the activity of a mitotic kinase such as TTK protein kinase.
  • Peptide fragments may be generated wholly or partly by chemical synthesis that block the catalytic sites of the kinase.
  • Peptide fragments can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984 ), in M. Bodanzsky and A.
  • Bodanzsky The Practice of Peptide Synthesis, Springer Verlag, New York (1984 ); and Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California
  • they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.
  • candidate compounds for inhibiting a mitotic kinase may be based on modelling the 3-dimensional structure of these enzymes and using rational drug design to provide candidate compounds with particular molecular shape, size and charge characteristics.
  • a candidate inhibitor for example, may be a "functional analogue" of a peptide fragment or other compound which inhibits the component.
  • a functional analogue has the same functional activity as the peptide or other compound in question. Examples of such analogues include chemical compounds which are modelled to resemble the three dimensional structure of the component in an area which contacts another component, and in particular the arrangement of the key amino acid residues as they appear.
  • Another class of inhibitors useful for treatment of PTEN mutated or deficient cancer includes nucleic acid inhibitors of TTK protein kinase , or the complements thereof, which inhibit activity or function by down-regulating production of active polypeptide. This can be monitored using conventional methods well known in the art, for example by screening using real time PCR as described in the examples.
  • mitotic kinases may be inhibited using anti-sense or RNAi technology.
  • anti-sense or RNAi technology The use of these approaches to down-regulate gene expression is now well-established in the art.
  • Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of the base excision repair pathway component so that its expression is reduced or completely or substantially completely prevented.
  • anti-sense techniques may be used to target control sequences of a gene, e.g. in the 5' flanking sequence, whereby the anti-sense oligonucleotides can interfere with expression control sequences.
  • the construction of anti-sense sequences and their use is described for example in Peyman & Ulman, Chemical Reviews, 90:543-584, 1990 and Crooke, Ann. Rev. Pharmacol. Toxicol., 32:329-376, 1992 .
  • Oligonucleotides may be generated in vitro or ex vivo for administration or anti-sense RNA may be generated in vivo within cells in which down-regulation is desired.
  • double-stranded DNA may be placed under the control of a promoter in a "reverse orientation" such that transcription of the anti-sense strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the sense strand of the target gene.
  • the complementary anti-sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein. Whether or not this is the actual mode of action is still uncertain. However, it is established fact that the technique works.
  • the complete sequence corresponding to the coding sequence in reverse orientation need not be used.
  • fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding or flanking sequences of a gene to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon.
  • a suitable fragment may have about 14-23 nucleotides, e.g., about 15, 16 or 17 nucleotides.
  • RNAi RNA interference
  • RNA interference is a two-step process.
  • dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs ( ⁇ 2nt).
  • siRNAs target the corresponding mRNA sequence specifically for destruction ( Zamore, Nature Structural Biology, 8, 9, 746-750, 2001 .
  • RNAi may also be efficiently induced using chemically synthesized siRNA duplexes of the same structure with 3'-overhang ends ( Zamore et al, Cell, 101: 25-33, 2000 ). Synthetic siRNA duplexes have been shown to specifically suppress expression of endogenous and heterologeous genes in a wide range of mammalian cell lines ( Elbashir et al, Nature, 411: 494-498, 2001 ).
  • nucleic acid is used which on transcription produces a ribozyme, able to cut nucleic acid at a specific site and therefore also useful in influencing gene expression, e.g., see Kashani-Sabet & Scanlon, Cancer Gene Therapy, 2(3): 213-223, 1995 and Mercola & Cohen, Cancer Gene Therapy, 2(1): 47-59, 1995 .
  • Small RNA molecules may be employed to regulate gene expression. These include targeted degradation of mRNAs by small interfering RNAs (siRNAs), post transcriptional gene silencing (PTGs), developmentally regulated sequence-specific translational repression of mRNA by micro-RNAs (miRNAs) and targeted transcriptional gene silencing.
  • siRNAs small interfering RNAs
  • PTGs post transcriptional gene silencing
  • miRNAs micro-RNAs
  • targeted transcriptional gene silencing targeted transcriptional gene silencing.
  • Double-stranded RNA (dsRNA)-dependent post transcriptional silencing also known as RNA interference (RNAi)
  • RNAi Double-stranded RNA
  • RNAi RNA interference
  • a 20-nt siRNA is generally long enough to induce gene-specific silencing, but short enough to evade host response. The decrease in expression of targeted gene products can be extensive with 90% silencing induced by a few molecules of siRNA.
  • RNA sequences are termed “short or small interfering RNAs” (siRNAs) or “microRNAs” (miRNAs) depending on their origin. Both types of sequence may be used to down-regulate gene expression by binding to complimentary RNAs and either triggering mRNA elimination (RNAi) or arresting mRNA translation into protein.
  • siRNA are derived by processing of long double stranded RNAs and when found in nature are typically of exogenous origin.
  • Micro-interfering RNAs are endogenously encoded small non-coding RNAs, derived by processing of short hairpins. Both siRNA and miRNA can inhibit the translation of mRNAs bearing partially complimentary target sequences without RNA cleavage and degrade mRNAs bearing fully complementary sequences.
  • the siRNA ligands are typically double stranded and, in order to optimise the effectiveness of RNA mediated down-regulation of the function of a target gene, it is preferred that the length of the siRNA molecule is chosen to ensure correct recognition of the siRNA by the RISC complex that mediates the recognition by the siRNA of the mRNA target and so that the siRNA is short enough to reduce a host response.
  • miRNA ligands are typically single stranded and have regions that are partially complementary enabling the ligands to form a hairpin.
  • miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein. A DNA sequence that codes for a miRNA gene is longer than the miRNA. This DNA sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement base pair to form a partially double stranded RNA segment.
  • the design of microRNA sequences is discussed in John et al, PLoS Biology, 11(2), 1862-1879, 2004 .
  • the RNA ligands intended to mimic the effects of siRNA or miRNA have between 10 and 40 ribonucleotides (or synthetic analogues thereof), more preferably between 17 and 30 ribonucleotides, more preferably between 19 and 25 ribonucleotides and most preferably between 21 and 23 ribonucleotides.
  • the molecule may have symmetric 3' overhangs, e.g. of one or two (ribo)nucleotides, typically a UU of dTdT 3' overhang.
  • siRNA and miRNA sequences can be synthetically produced and added exogenously to cause gene downregulation or produced using expression systems (e.g. vectors).
  • expression systems e.g. vectors
  • the siRNA is synthesized synthetically.
  • Longer double stranded RNAs may be processed in the cell to produce siRNAs (e.g. see Myers, Nature Biotechnology, 21: 324-328, 2003 ).
  • the longer dsRNA molecule may have symmetric 3' or 5' overhangs, e.g. of one or two (ribo)nucleotides, or may have blunt ends.
  • the longer dsRNA molecules may be 25 nucleotides or longer.
  • the longer dsRNA molecules are between 25 and 30 nucleotides long. More preferably, the longer dsRNA molecules are between 25 and 27 nucleotides long. Most preferably, the longer dsRNA molecules are 27 nucleotides in length.
  • dsRNAs 30 nucleotides or more in length may be expressed using the vector pDECAP ( Shinagawa et al., Genes and Dev., 17: 1340-5, 2003 ).
  • shRNAs are more stable than synthetic siRNAs.
  • a shRNA consists of short inverted repeats separated by a small loop sequence. One inverted repeat is complimentary to the gene target.
  • the shRNA is processed by DICER into a siRNA which degrades the target gene mRNA and suppresses expression.
  • the shRNA is produced endogenously (within a cell) by transcription from a vector.
  • shRNAs may be produced within a cell by transfecting the cell with a vector encoding the shRNA sequence under control of a RNA polymerase III promoter such as the human H1 or 7SK promoter or a RNA polymerase II promoter.
  • the shRNA may be synthesised exogenously (in vitro) by transcription from a vector.
  • the shRNA may then be introduced directly into the cell.
  • the shRNA sequence is between 40 and 100 bases in length, more preferably between 40 and 70 bases in length.
  • the stem of the hairpin is preferably between 19 and 30 base pairs in length.
  • the stem may contain G-U pairings to stabilise the hairpin structure.
  • the siRNA, longer dsRNA or miRNA is produced endogenously (within a cell) by transcription from a vector.
  • the vector may be introduced into the cell in any of the ways known in the art.
  • expression of the RNA sequence can be regulated using a tissue specific promoter.
  • the siRNA, longer dsRNA or miRNA is produced exogenously ( in vitro ) by transcription from a vector.
  • siRNA molecules may be synthesized using standard solid or solution phase synthesis techniques, which are known in the art.
  • Linkages between nucleotides may be phosphodiester bonds or alternatives, e.g., linking groups of the formula P(O)S, (thioate); P(S)S, (dithioate); P(O)NR'2; P(O)R'; P(O)OR6; CO; or CONR'2 wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotides through-O-or-S-.
  • Modified nucleotide bases can be used in addition to the naturally occurring bases, and may confer advantageous properties on siRNA molecules containing them.
  • modified bases may increase the stability of the siRNA molecule, thereby reducing the amount required for silencing.
  • the provision of modified bases may also provide siRNA molecules, which are more, or less, stable than unmodified siRNA.
  • modified nucleotide base' encompasses nucleotides with a covalently modified base and/or sugar.
  • modified nucleotides include nucleotides having sugars, which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3'position and other than a phosphate group at the 5'position.
  • modified nucleotides may also include 2'substituted sugars such as 2'-O-methyl- ; 2-0-alkyl ; 2-0-allyl ; 2'-S-alkyl; 2'-S-allyl; 2'-fluoro- ; 2'-halo or 2; azido-ribose, carbocyclic sugar analogues ⁇ -anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars and sedoheptulose.
  • 2'substituted sugars such as 2'-O-methyl- ; 2-0-alkyl ; 2-0-allyl ; 2'-S-alkyl; 2'-S-allyl; 2'-fluoro- ; 2'-halo or 2; azido-ribose, carbocyclic sugar analogues ⁇ -anomeric sugars; epimeric sugars such as arabi
  • Modified nucleotides include alkylated purines and pyrimidines, acylated purines and pyrimidines, and other heterocycles. These classes of pyrimidines and purines are known in the art and include pseudoisocytosine, N4,N4-ethanocytosine, 8-hydroxy-N6-methyladenine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil, 5 fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl uracil, dihydrouracil, inosine, N6-isopentyl-adenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 2,2-dimethylguanine, 2methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylgu
  • the present invention is concerned with methods of screening candidate compounds to determine whether one or more candidate agents are likely to be useful for the treatment of PTEN mutated or deficient cancer.
  • methods of screening candidate compounds to determine whether one or more candidate agents are likely to be useful for the treatment of PTEN mutated or deficient cancer.
  • PTEN mutant cell line it may be advantageous to use a PTEN mutant cell line to test the effectiveness of a candidate TTK protein kinase inhibitor.
  • a full list of PTEN mutant cell lines is available for retrieved from the COSMIC database:
  • a method of screening may involve using cell lines to determine whether a candidate agent is synthetically lethal in a first cell line which is PTEN mutated or deficient.
  • This method preferably also uses a second cell line that is PTEN proficient as a control and candidate agents are selected which are synthetically lethal in the first cell line and which preferably do not cause any substantial amount cell death in the second cell line and/or normal cells.
  • synthetic lethality in cancer cells. Two mutations are synthetically lethal if cells with either of the single mutations are viable, but cells with both mutations are inviable. Identifying synthetic lethal combinations therefore allows a distinct approach to identifying therapeutic targets that allows selective killing of tumour cells.
  • the method is carried out using cancer cell lines, e.g. mammalian or human cancer cell lines, and more specifically PTEN mutated or deficient cancer cell lines.
  • RNAi screens One preferred way of initially identifying synthetic lethal interactions involves the use of RNAi screens.
  • Synthetic lethality describes the scenario in which two normally non-essential genes become essential when both are lost, or inhibited.
  • Targeting a gene that is synthetically lethal with a cancer specific mutation should selectively kill tumour cells while sparing normal cells.
  • One of the major advantages of this approach is the ability to target cancer cells containing loss-of-function mutations, that is, mutations in tumour suppressor genes.
  • loss-of-function mutations that is, mutations in tumour suppressor genes.
  • Most pharmacological agents inhibit rather than activate protein function and therefore cannot be used to target loss-of-function alterations in tumours. Identification of synthetic lethal relationships with tumour suppressor genes could allow cells that contain the tumour suppressor mutations to be selectively killed.
  • RNAi screens it is now possible, in principle, to perform large-scale synthetic-lethal gene identification in mammalian cells, as is routinely done in yeast. Screening deletion mutants that have defects in cell-cycle checkpoint or DNA repair mechanisms in yeast has yielded synthetically lethal genes and small-molecule inhibitors. Using mammalian isogenic-paired cell lines that differ in a single genetic target, RNAi can be used to identify drug targets that when inhibited will result in the selective death of tumour cells.
  • the present invention also includes methods of screening that employ mitotic kinases as protein targets for the screening of candidate compounds to find mitotic kinase inhibitors. Accordingly, methods of screening may be carried out for identifying candidate agents that are capable of inhibiting TTK protein kinase for subsequent use of development as agents for the treatment of PTEN mutated or deficient cancer. Conveniently, this may be done in an assay buffer to help the components of the assay interact, and in a multiple well format to test a plurality of candidate agents.
  • the activity of a mitotic kinase can then be determined in the presence and absence of the one or more candidate compounds to determine whether a given candidate is an inhibitor of a mitotic kinase.
  • the candidate agent may be a known inhibitor of one of the protein targets disclosed herein, an antibody, a peptide, a nucleic acid molecule or an organic or inorganic compound, e.g. molecular weight of less than 100 Da.
  • candidate agents that are compounds are preferred.
  • combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a target protein.
  • Such libraries and their use are known in the art.
  • the present invention also specifically envisages screening candidate agents known for the treatment of other conditions, and especially other forms of cancer. This has the advantage that the patient or disease profile of known therapeutic agents might be expanded or modified using the screening techniques disclosed herein, or for therapeutic agents in development, patient or disease profiles established that are relevant for the treatment of PTEN mutated or deficient cancer.
  • the agent in question may be tested to determine whether it is not lethal to normal cells or otherwise is suited to therapeutic use. Following these studies, the agent may be manufactured and/or used in the preparation of a medicament, pharmaceutical composition or dosage form.
  • peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
  • Mimetic design, synthesis and testing is generally used to avoid randomly screening large number of molecules for a target property.
  • the pharmacophore Once the pharmacophore has been found, its structure is modelled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process. In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.
  • the physical properties e.g. stereochemistry, bonding, size and/or charge
  • data from a range of sources e.g. spectroscopic techniques, X-ray diffraction data and NMR.
  • Computational analysis, similarity mapping
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • a cancer may be identified as PTEN mutated or deficient cancer by testing a sample of cancer cells from an individual, for example to determine whether the PTEN protein contains one or more mutations or to determine the expression of the PTEN gene to evaluate whether expression of the protein is absent or at a reduced level compared to normal. It is known that genetic inactivation of PTEN occurs in glioblastoma, endometrial cancer and prostate cancer and reduced expression is found in many other tumor types that include lung and breast cancer.
  • Examples of known PTEN mutated or deficient cancers that are treatable in accordance with the present invention include cancers affecting the autonomic ganglia, biliary tract, bone, breast, CNS, cervix, endometrium, eye, haematopoietic and lymphoid tissue, kidney, large intestine, liver, lung, meninges, oesophagus, ovary, pancreas, prostate, salivary gland, skin, soft tissue, stomach, testis, thyroid, upper aerodigestive tract, urinary tract, or vulva.
  • Cancer associated mutations in PTEN occur across the entire coding sequence of the gene and include both premature truncating mutations and single amino acid substitutions in both the catalytic and non-catalytic domains of the protein.
  • the frequency of monoallelic mutations in PTEN has been estimated at 50%-80% in sporadic tumors (including endometrial carcinoma, glioblastoma, and prostate cancer) and at 30%-50% in breast, colon, and lung tumors.
  • Complete loss of PTEN is observed at highest frequencies in endometrial cancer and glioblastoma and is generally associated with advanced cancers and metastases.
  • Common tumor associated PTEN mutations include c.388C>G, c.697C>T, c.388C>T, c.800delA, c.968delA, c.517C>T, c.968_969insA, c.518G>A, c.955_958delACTT, c.1003C>T, c.950_953delTACT.
  • the sample may be of normal cells from the individual where the individual has a mutation in the PTEN gene or the sample may be of cancer cells, e.g. where the cells forming a tumour contain one or more PTEN mutations.
  • Activity may be determined relative to a control, for example in the case of defects in cancer cells, relative to non-cancerous cells, preferably from the same tissue.
  • the activity of the PTEN may be determined by using techniques well known in the art such as Western blot analysis, immunohistology, chromosomal abnormalities, enzymatic or DNA binding assays and plasmid-based assays.
  • the determination of PTEN gene expression may involve determining the presence or amount of PTEN mRNA in a sample. Methods for doing this are well known to the skilled person. By way of example, they include determining the presence of PTEN mRNA (i) using a labelled probe that is capable of hybridising to the PTEN nucleic acid; and/or (ii) using PCR involving one or more primers based on a PTEN nucleic acid sequence to determine whether the PTEN transcript is present in a sample.
  • the probe may also be immobilised as a sequence included in a microarray.
  • detecting PTEN mRNA is carried out by extracting RNA from a sample of the tumour and measuring PTEN expression specifically using quantitative real time RT-PCR.
  • the expression of PTEN could be assessed using RNA extracted from a tumour sample using microarray analysis, which measures the levels of mRNA for a group of genes using a plurality of probes immobilised on a substrate to form the array.
  • the determination of whether a patient has a PTEN mutated or deficient cancer can be carried out by analysis of PTEN protein expression, for example to examining whether reduced levels of PTEN protein are expressed or whether the PTEN protein contains one or more mutations.
  • the presence or amount of PTEN protein may be determined using a binding agent capable of specifically binding to the PTEN protein, or fragments thereof.
  • a preferred type of PTEN protein binding agent is an antibody capable of specifically binding the PTEN or fragment thereof.
  • the antibody may be labelled to enable it to be detected or capable of detection following reaction with one or more further species, for example using a secondary antibody that is labelled or capable of producing a detectable result, e.g. in an ELISA type assay.
  • a labelled binding agent may be employed in a western blot to detect PTEN protein.
  • the method for determining the presence of PTEN protein may be carried out on tumour samples, for example using immunohistochemical (IHC) analysis.
  • IHC analysis can be carried out using paraffin fixed samples or frozen tissue samples, and generally involves staining the samples to highlight the presence and location of PTEN protein.
  • the active agents disclosed herein for the treatment of PTEN mutated or deficient cancer may be administered alone, but it is generally preferable to provide them in pharmaceutical compositions that additionally comprise with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents.
  • pharmaceutical compositions are provided in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins .
  • small molecule therapeutics useful for treating PTEN mutated or deficient cancer include: BEZ235, Olaparib and GDC0941.
  • derivatives of the therapeutic agents includes salts, coordination complexes, esters such as in vivo hydrolysable esters, free acids or bases, hydrates, prodrugs or lipids, coupling partners.
  • Salts of the compounds of the invention are preferably physiologically well tolerated and non toxic. Many examples of salts are known to those skilled in the art.
  • Compounds having acidic groups such as phosphates or sulfates, can form salts with alkaline or alkaline earth metals such as Na, K, Mg and Ca, and with organic amines such as triethylamine and Tris (2-hydroxyethyl)amine.
  • Salts can be formed between compounds with basic groups, e.g., amines, with inorganic acids such as hydrochloric acid, phosphoric acid or sulfuric acid, or organic acids such as acetic acid, citric acid, benzoic acid, fumaric acid, or tartaric acid.
  • Compounds having both acidic and basic groups can form internal salts.
  • Esters can be formed between hydroxyl or carboxylic acid groups present in the compound and an appropriate carboxylic acid or alcohol reaction partner, using techniques well known in the art.
  • Derivatives which as prodrugs of the compounds are convertible in vivo or in vitro into one of the parent compounds.
  • at least one of the biological activities of compound will be reduced in the prodrug form of the compound, and can be activated by conversion of the prodrug to release the compound or a metabolite of it.
  • Coupled derivatives include coupling partners of the compounds in which the compounds is linked to a coupling partner, e.g. by being chemically coupled to the compound or physically associated with it.
  • Examples of coupling partners include a label or reporter molecule, a supporting substrate, a carrier or transport molecule, an effector, a drug, an antibody or an inhibitor.
  • Coupling partners can be covalently linked to compounds of the invention via an appropriate functional group on the compound such as a hydroxyl group, a carboxyl group or an amino group.
  • Other derivatives include formulating the compounds with liposomes.
  • pharmaceutically acceptable includes compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a subject e.g. human
  • Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • the active agents disclosed herein for the treatment of PTEN mutated or deficient cancer according to the present invention are preferably for administration to an individual in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual.
  • a prophylaxis may be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, Lippincott, Williams & Wilkins .
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing the active compound into association with a carrier, which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • the agents disclosed herein for the treatment of PTEN mutated or deficient cancer may be administered to a subject by any convenient route of administration, whether systemically/ peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g.
  • vaginal parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly.
  • Formulations suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • Suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.
  • concentration of the active compound in the solution is from about 1 ng/ml to about 10 ⁇ g/ml, for example from about 10 ng/ml to about 1 ⁇ g/ml.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs.
  • compositions comprising agents disclosed herein for the treatment PTEN mutated or deficient cancer may be used in the methods described herein in combination with standard chemotherapeutic regimes or in conjunction with radiotherapy.
  • chemotherapeutic agents include Amsacrine (Amsidine), Bleomycin, Busulfan, Capecitabine (Xeloda), Carboplatin, Carmustine (BCNU), Chlorambucil(Leukeran), Cisplatin, Cladribine(Leustat), Clofarabine (Evoltra), Crisantaspase (Erwinase), Cyclophosphamide, Cytarabine (ARA-C), dacarbazine (DTIC), Dactinomycin (Actinomycin D),Daunorubicin, Docetaxel (Taxotere), Doxorubicin, Epirubicin, Etoposide (Vepesid, VP-16), Fludarabine (Fludara), Fluorouracil (5-FU),
  • Ifosfamide (Mitoxana), Irinotecan (CPT-11, Campto), Leucovorin (folinic acid), Liposomal doxorubicin (Caelyx, Myocet), Liposomal daunorubicin (DaunoXome®) Lomustine, Melphalan, Mercaptopurine, Mesna, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin (Eloxatin), Paclitaxel (Taxol), Pemetrexed (Alimta), Pentostatin (Nipent), Procarbazine, Raltitrexed (Tomudex®), Streptozocin (Zanosar®), Tegafur-uracil (Uftoral), Temozolomide (Temodal), Teniposide (Vumon), Thiotepa, Tioguanine (6-TG) (Lanvis), Topotecan (Hycamtin), Treosulfan, Vinblast
  • Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.
  • a suitable dose of the active compound is in the range of about 100 ⁇ g to about 250 mg per kilogram body weight of the subject per day.
  • the active compound is a salt, an ester, prodrug, or the like
  • the amount administered is calculated on the basis of the parent compound, and so the actual weight to be used is increased proportionately.
  • RNAiMAX RNAiMAX transfection reagents.
  • the siRNA library siARRAY - targeting 714 known and putative human protein kinase genes was obtained in nine 96 well plates from Dharmacon (USA). Each well in this library contained a SMARTpool of four distinct siRNA species targeting different sequences of the target transcript. Each plate was supplemented with siCONTROL (ten wells, Dharmacon (USA)).
  • Antibodies targeting the following were used as per manufacturers instructions: ACTIN and C-MYC (Santa Cruz Biotech), TTK, ER, PR, ERBB2, CYCLIN D1, TFF1, FOXO1, C-JUN, and PTEN C-terminus (Cell Signalling, Danvers, USA). All secondary antibodies used for western blot analysis were HRP conjugated.
  • Protein lysates were prepared using RIPA lysis buffer (50 nM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.1% DOC, 1% TritonX-100, 50 mM NaF, 1 mM Na 3 VO 4 and protease inhibitors). 100mg of total cell lysate was loaded onto prefabricated 4-12% Bis-Tris gels (Invitrogen), with full range rainbow molecular weight marker (GE Healthcare, UK) as a size reference, and resolved by SDS-PAGE electrophoresis. Proteins were transferred to nitrocellulose membrane (Bio-rad, USA), blocked and probed with primary antibody diluted 1 in 1000 in 5% milk overnight at 4°C. Secondary antibodies were diluted 1 in 5000 in 5% milk and incubated for one hour at room temperature. Protein bands were visualised using ECL (GE Healthcare, UK) and MR or XAR film (Kodak).
  • RNAi gene silencing was determined by western blotting and by viability assays of silencing effects with individual oligos.
  • Cells were transfected with individual ERBB2, ESR1, PIK3CA or TTK siGenome oligos (Dharmacon). Protein lysates were collected 48 hours following transfection for western blot analysis. Cell viability was measured using CellTiter Glo (Promega, USA) after 5 populations doublings.
  • HCT116 PTEN wt or PTEN null cells were treated with colcemide (10ng/ml, Sigma) and MG132 (20uM, Sigma) for 1 hour. Cells were then lysed in hypotonic solution (0.03M Sodium Citrate) for 20 minutes at 37°C and fixed in methanol/acetic acid (3:1). Two or three drops of suspended cells were applied to glass slides and chromosomes were stained with DAPI.
  • LSI PTEN (10q23, red/orange) /chromosome 10 centromere (CEP 10, green) Dual Colour Probe (Abbott Molecular, IL, US) was hybridised to representative slides of the cell lines according to the manufacturer's instructions. Signals were counted in 100 non-overlapping nuclei using the Leica TCS SP2 confocal microscope (Leica, Milton Keynes, UK).
  • cRNA samples were hybridised on Illumina human-6 v2 BeadChips, covering approximately 47,000 RefSeq transcripts.
  • the random distribution of large populations of oligonucleotide-coated beads across the available positions within the human-6 v2 chip enables, on average, 30 intensity measurements per RefSeq, yielding quantitative assessments of gene expression.
  • All basic expression data analysis was carried out using the manufacturer's software BeadStudio 3.1.
  • Illumina expression profiles were performed in triplicate, the raw data were then variance-stabilizing transformed and robust spine normalised using the lumi package in the Bioconductor software. Expression values for each sample were median scaled and the mean expression value was established over the three replicates. Genes with significant difference in expression between cell lines were identified by one-way analysis of variance (ANOVA).
  • Genomic DNA was extracted from cell lines using the QIAamp DNA Blood Mini Kit (51104, Qiagen), according to manufacturer's instructions.
  • Microarray-based CGH analysis was performed on an in-house 32K tiling path BAC array platform as previously described (29).
  • CBS circular binary segmentation
  • AWS smoothed log2 ratio values ⁇ -0.12 were categorised as losses, those >0.12 as gains, and those in between as unchanged. Amplifications were defined as smoothed log2 ratio values >0.4 (29).
  • aCGH and gene expression were compared by direct Pearson correlation of gene expression log intensity values with smoothed log ratio values for every probe in the gene expression data. Correlation p values were adjusted for multiple comparison testing using the local False Discovery Rate (FDR) method of Benjamini and Hochberg (30) as previously described (28). Mann Whitney U tests were preformed for each gene to compare gene expression values in groups defined as assigned as amplified or not, gained or not, lost or not and deleted or not using the thresholded aCGH calls. Wilcox test p-values were corrected for multiple comparison testing within contiguously altered regions. For each gene, cases in which genes were amplified, gained, lost or deleted were recorded along with the fold changes between samples carrying a copy number change and those which did not. Total counts of gains, losses, amplifications and deletions were also recorded.
  • FDR False Discovery Rate
  • siRNA Z score Correlation of siRNA Z score with gene expression and aCGH data The correlation between siRNA Z score and normalised gene expression was examined for genes where siRNA caused significant loss of viability (Z ⁇ -2). Z score was compared to normalized gene expression using Pearson correlation coefficient. A gene was taken as being significantly correlated if the Pearson correlation coefficient was significantly different to the null hypothesis, the correlation was inverse, and the variation in gene expression between cells lines were significantly different as assessed by one-way ANOVA.
  • STR short tandem repeat
  • siRNA RNA interference/short interfering RNA
  • each cell line was transfected with a 96 well-plate arrayed siRNA library targeting 714 kinases and kinase-related genes (see Methods). After five population doublings, cell viability in each well was estimated by use of a highly sensitive luminescent assay measuring cellular ATP levels. To identify loss of viability /inhibition/failure to proliferate effects in each cell line, luminescence readings from each well were log transformed and then centred by the plate median, to account for plate-to-plate variation.
  • RNA interference screens were carried out in triplicate and comparison of Z score data from replica screens of each cell line showed the screening process to be highly robust.
  • This panel encapsulated each of the major breast cancer subtypes (4) and included hormone receptor (Estrogen Receptor (ER), Progesterone Receptor (PR)), ERBB2 positive as well as ER, PR and ERBB2 negative models (triple negative), as characterised by parallel immunohistochemical, fluorescent in situ hybridisation (FISH) and western blot analysis.
  • FISH fluorescent in situ hybridisation
  • the delineation of functional viability profiles for the breast cell line panel provided a framework for identifying dependencies in particular genetically-defined subgroups of the disease.
  • Somatic mutations of PIK3CA which encodes the p110 ⁇ catalytic subunit of PI3-kinase, have been shown to induce oncogenic transformation in vitro and in vivo (6).
  • PIK3CA mutations are found in 8% - 35% of human breast cancers making them one of the most common genetic aberrations in this disease (7).
  • PIK3CA mutant breast cancer lines were preferentially sensitive to AKT2 and AKT3 siRNAs ( Fig. 1c ).
  • mutant PIK3CA mediates cellular transformation Although the mechanisms by which mutant PIK3CA mediates cellular transformation are not completely understood, it is likely that part of this effect is mediated by signalling through AKT (8).
  • the preferential sensitivity of PIK3CA mutant cells to AKT targeting supported the hypothesis that the viability profiles had the ability to illuminate true addiction pathways.
  • helical and kinase domain mutants have distinct physiologic phenotypes in human cells (9, 10), and the differential effects of PIK3CA targeting in helical vs. kinase domain mutants could also suggest differences in PIK3CA addiction.
  • PTEN deficient breast tumor cells are dependent upon the mitotic checkpoint kinase TTK
  • TTK inhibition may be a novel therapeutic strategy for treating PTEN mutant tumours. Aneuploidy is frequently observed in both human breast carcinomas with low expression of PTEN and prostatic intraepithelial neoplasia from Pten mutant mice (17). This latter phenomenon is perhaps explained by the centromeric dysfunction in PTEN mutant tumor cells, most likely mediated by a loss of the interaction between PTEN and CENP-C, a key kinetochore component (18).
  • TTK is required for normal function of the mitotic spindle checkpoint and it is established that TTK inhibition drives early exit from mitosis and chromosomal aneuploidy. In tumor cells with an aneuploid phenotype TTK inhibition further exacerbates aneupliody and is particularly lethal (19).
  • Functional viability profiling identifies candidate functional taxonomies
  • Hierarchical cluster analysis of transcript profiles classifies breast tumours and tumor cell lines into luminal and basal-like molecular subtypes (14).
  • Hierarchical clustering of genes that when silenced caused loss of viability (Z ⁇ -2) in two or more cell lines revealed two distinct groups, distinct from those formed by the clustering of the expression data ( Fig. 3 .
  • CAL51 and MDAMB453, which carry both PTEN and PIK3CA mutations were classified into Group 1.
  • the ERBB2 amplified cell lines were distributed evenly between the two groups, the cell lines resistant to ERBB2 silencing and also lapatinib treatment were all contained within Group 1 (JIMT1, MDAMB453 and VP229) and those sensitive to ERBB2 silencing/lapatinib (HCC202, BT474 and SKBR3) fell into Group 2 ( Fig. 3b ).
  • the distinction between Groups 1 and 2 implies that our panel of breast cancer cell lines functionally divide into two groups according to their dependency on well-established essential cancer networks, and is independent from the currently used clinical and transcriptomically-defined breast cancer subgroups.

Claims (17)

  1. TTK-Proteinkinase-Inhibitor zur Verwendung in einem Behandlungsverfahren eines Individuums mit Krebs durch die Inhibierung der TTK-Proteinkinase, wobei der Krebs ein PTEN- (Phosphatase und Tensin-Homolog) mutierter oder -defizienter Krebs ist.
  2. TTK-Proteinkinase-Inhibitor zur Verwendung in einem Behandlungsverfahren nach Anspruch 1, wobei der PTEN-defiziente Krebs PTEN-Null ist oder der PTEN-mutierte Krebs eine trunkierende Mutation oder eine oder mehrere Substitutionen umfasst.
  3. TTK-Proteinkinase-Inhibitor zur Verwendung in einem Behandlungsverfahren nach einem der vorangegangenen Ansprüche, wobei der PTEN-mutierte oder -defiziente Krebs den/die/das autonomen Ganglien, Gallenwege, Knochen, Brust, ZNS, Gebärmutterhals, Endometrium, Auge, hämatopoetische und lymphoide Gewebe, Niere, Dickdarm, Leber, Lunge, Meninges, Ösophagus, Eierstock, Bauchspeicheldrüse, Prostata, Speicheldrüsen, Haut, Weichgewebe, Magen, Hoden, Schilddrüsen, oberen Aerodigestivtrakt, Harntrakt oder Vulva betrifft.
  4. TTK-Proteinkinase-Inhibitor zur Verwendung in einem Behandlungsverfahren nach einem der Ansprüche 1 bis 3, wobei der PTEN-mutierte oder -defiziente Krebs Brustkrebs, Endometriumkarzinom, Glioblastom, Prostatakrebs, Darmkrebs oder Lungenkrebs ist.
  5. TTK-Proteinkinase-Inhibitor zur Verwendung in einem Behandlungsverfahren nach einem der vorangegangenen Ansprüche, wobei:
    (a) der Inhibitor ein Nucleinsäureinhibitor, ein Antikörper, ein kleines Molekül wie z.B. AZ3146 oder CCT132774 oder ein Peptid ist; oder
    (b) der Inhibitor ein Nucleinsäureinhibitor ist, der ein RNAi-Molekül oder ein siRNA-Molekül oder ein shRNA-Molekül ist.
  6. TTK-Proteinkinase-Inhibitor zur Verwendung in einem Behandlungsverfahren nach einem der vorangegangenen Ansprüche, wobei die Behandlung mit einem mitotischen Kinaseinhibitor mit einer weiteren Antikrebstherapie kombiniert wird, wobei die Behandlung mit dem mitotischen Kinaseinhibitor gegebenenfalls in Verbindung mit einem weiteren Chemotherapeutikum verwendet wird
  7. TTK-Proteinkinase-Inhibitor zur Verwendung in einem Behandlungsverfahren nach Anspruch 6, wobei das weitere Chemotherapeutikum Amsacrin (Amsidin), Bleomycin, Busulfan, Capecitabin (Xeloda), Carboplatin, Carmustin (BCNU), Chlorambucil (Leukeran), Cisplatin, Cladribin (Leustat), Clofarabin (Evoltra), Crisantaspase (Erwinase), Cyclophosphamid, Cytarabin (ARA-C), Dacarbazin (DTIC), Dactinomycin (Actinomycin D), Daunorubicin, Docetaxel (Taxotere), Doxorubicin, Epirubicin, Etoposid (Vepesid, VP-16), Fludarabin (Fludara), Fluoruracil (5-FU), Gemcitabin (Gemzar), Hydroxyharnstoff (Hydroxycarbamid, Hydrea), Idarubicin (Zavedos), Ifosfamid (Mitoxana), Irinotecan (CPT-11, Campto), Leucovorin (Folinsäure), Liposomales Doxorubicin (Caelyx, Myocet), Liposomales Daunorubicin (DaunoXome®), Lomustin, Melphalan, Mercaptopurin, Mesna, Methotrexat, Mitomycin, Mitoxantron, Oxaliplatin (Eloxatin), Paclitaxel (Taxol), Pemetrexed (Alimta), Pentostatin (Nipent), Procarbazin, Raltitrexed (Tomudex®), Streptozocin (Zanosar®), Tegafur-Uracil (Uftoral), Temozolomid (Temodal), Teniposid (Vumon), Thiotepa, Tioguanin (6-TG) (Lanvis), Topotecan (Hycamtin), Treosulfan, Vinblastin (Velbe), Vincristin (Oncovin), Vindesin (Eldisin) oder Vinorelbin (Navelbin) ist.
  8. TTK-Proteinkinase-Inhibitor zur Verwendung in einem Behandlungsverfahren nach Anspruch 6 oder Anspruch 7, wobei das weitere Chemotherapeutikum 5FU, ein BCL-XL-Inhibitor, ein BCR-ABL-, cKIT- oder PDGFR-Inhibitor, ein CDK-Inhibitor, ein CHK-Inhibitor, ein COX2-Inhibitor, ein EGFR-Inhibitor, ein gegen HER2 gerichtetes Mittel, ein HSP-Inhibitor, ein hTERT-Inhibitor, an IDO-Inhibitor, ein gegen MDM2 gerichtetes Mittel, Methotrexat, ein mTOR-Inhibitor, ein PARP-Inhibitor, ein PI3K-Inhibitor, ein Platinsalz, ein Proteasom-Inhibitor, ein RAR- oder RXR-Inhibitor, ein SRC-Inhibitor, ein TGFB2-Inhibitor, ein Topoisomerase-Inhibitor, ein VEGF-, RAF-, cKIT- oder PDGFR-Inhibitor oder ein SMC-Mimetikum ist.
  9. TTK-Proteinkinase-Inhibitor zur Verwendung in einem Behandlungsverfahren nach einem der vorangegangenen Ansprüche, wobei das Verfahren den Schritt des Bestimmens, ob das Individuum einen PTEN-mutierten oder -defizienten Krebs aufweist, umfasst.
  10. TTK-Proteinkinase-Inhibitor zur Verwendung in einem Behandlungsverfahren nach einem der vorangegangenen Ansprüche, wobei das Bestimmen, ob das Individuum PTEN-mutierten oder -defizienten Krebs aufweist, das Messen der PTEN-Proteinexpression in einer von dem Individuum erhaltenen Probe umfasst, um zu bestimmen, ob das PTEN-Protein mutiert oder defizient ist;
    und wobei gegebenenfalls das Bestimmen der PTEN-Proteinexpression eines oder mehrere aus dem Bestimmen der PTEN-Proteinexpression in einer Tumorprobe unter Verwendung von Immunhistochemie, Bestimmen der PTEN-Proteinexpression umfassend das Messen der PTEN-Proteinspiegel in einem Zelllysat mittels ELISA oder Western-Blot und/oder Bestimmen der PTEN-Proteinexpression umfassend die Verwendung eines Bindemittels, das in der Lage ist, spezifisch an das PTEN-Protein oder ein Fragment davon zu binden, umfasst.
  11. TTK-Proteinkinase-Inhibitor zur Verwendung in einem Behandlungsverfahren nach einem der vorangegangenen Ansprüche, wobei das Bestimmen, ob das Individuum PTEN-mutierten oder -defizienten Krebs aufweist, auf einer genomischen Nucleinsäure durchgeführt wird, die aus einer Probe von Zellen, die aus dem Brustkrebs erhalten wurden, oder aus einer Probe von Krebszellen, die im Blut zirkulieren, extrahiert wurde;
    und wobei gegebenenfalls das Bestimmen der Expression des PTEN-Gens das Extrahieren von RNA aus einer Probe von Krebszellen und das Messen der Expression mittels Echtzeit-PCR und/oder unter Verwendung einer Sonde, die in der Lage ist, an PTEN-RNA zu hybridisieren, umfasst, wobei die Sonde gegebenenfalls in einem Mikroarray immobilisiert wird.
  12. TTK-Proteinkinase-Inhibitor zur Verwendung in einem Behandlungsverfahren nach einem der vorangegangenen Ansprüche, wobei das Verfahren den Ausgangsschritt des Erhaltens einer Probe von dem Individuum umfasst, wobei die Probe gegebenenfalls eine Tumorprobe, eine Blutprobe, eine Gewebeprobe oder eine Zellprobe ist.
  13. Verfahren zum Screening auf Mittel, die in der Behandlung eines PTEN-mutierten oder -defizienten Krebses nützlich sind, wobei das Verfahren erste und zweite Zelllinien verwendet, wobei die erste Zelllinie PTEN ist und die zweite Zelllinie PTENkompetent ist, wobei das Verfahren Folgendes umfasst:
    (a) Kontaktieren der ersten und zweiten Säugetierzelllinie mit zumindest einem Kandidatenmittel;
    (b) Bestimmen der Menge an Zelltod in der ersten und zweiten Zelllinie;
    (c) Auswählen eines Kandidatenmittels, das in der ersten Zelllinie synthetisch tödlich ist; und
    (d) Bestimmen, ob das in Schritt (c) ausgewählte Kandidatenmittel ein TTK-Proteinkinase-Inhibitor ist.
  14. Verfahren zum Screening auf Mittel, die in der Behandlung eines PTEN-mutierten oder -defizienten Krebses nützlich sind, wobei das Verfahren Folgendes umfasst:
    (a) Kontaktieren einer TTK-Proteinkinase mit zumindest einem Kandidatenmittel;
    (b) Bestimmen einer Wirkung des zumindest einen Kandidatenmittels auf eine Aktivität der TTK-Proteinkinase; und
    (c) Auswählen eines Kandidatenmittels, das die Aktivität der mitotischen Kinase hemmt, als nützlich für die Behandlung von PTEN-mutiertem oder -defizientem Krebs.
  15. Verfahren nach Anspruch 14, das weiters den Schritt des Kontaktierens eines Kandidatenmittels, das in Schritt (c) ausgewählt wurde, mit einer PTEN-mutierten oder -defizienten Krebszelllinie umfasst, um zu bestimmen, ob das Kandidatenmittel für die Krebszelllinie zytotoxisch ist.
  16. Verfahren nach Anspruch 14 oder Anspruch 15, wobei:
    (a) das Kandidatenmittel ein Kandidaten-Nucleinsäure-Inhibitor, ein Kandidaten-Antikörper oder ein kleines Kandidatenmolekül oder ein Kandidatenpeptid ist; und/oder
    (b) das Kandidatenmittel eine Verbindung ist, die Teil einer Verbindungsbibliothek ist, wobei die Kandidatenverbindung gegebenenfalls ein Molekulargewicht von weniger als 100 Da aufweist; und/oder
    (c) das/die Kandidatenmittel oder Kandidatenverbindungen ein Arzneimittel ist/sind, das/die für die Verwendung in der Behandlung von Krebs zugelassen ist/sind.
  17. Verfahren nach einem der Ansprüche 13 bis 16, das weiters das Bestimmen, ob ein Kandidatenmittel für normale Zellen nicht tödlich ist, und/oder das Bestimmen der Wirkung von Kombinationen aus zwei oder mehreren Kandidatenverbindungen auf die Zelllinien oder Protein-Targets umfasst.
EP12728113.7A 2011-06-10 2012-06-08 Materialien und verfahren zur behandlung von pten-mutiertem oder -defizientem krebs Not-in-force EP2718436B1 (de)

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